CZ283333B6 - O-alkylated derivatives of rapamycin, process of their preparation, pharmaceutical preparations containing thereof and their use, particularly as immunosuppressives - Google Patents

Abstract

Described are O-alkylated derivatives of rapamycin of the general formula I wherein X is (H, H) or O, Y is (H, OH) or O, R.sup.1. and R.sup.2.n. are independently selected from H, alkyl, thioalkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl, and the like. hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl, alkoxyalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxycarbonylaminoalkyl, acylaminoalkyl, arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl, dioxolanylallyl, carbalkoxyalkyl and (R.sup.3). .sup.3.n. is independently selected from H, methyl, ethyl, isopropyl, tert. butyl and phenyl, wherein "alk-" or "alkyl" is C 1-6 -alkyl, branched or straight, preferably C 1-6 -alkyl, in which it may be a carbon chain optionally interrupted by an ether (-O-) bond, and R.sup.4. is methyl or R 4 is 4. and R.sup.1.n. together form C.sub.2-6n.-alkylene, with the proviso that R.sup.1.n. and R.sup.2.n. are not both H and provided that when R.sup.1.n. is carbalkoxyalkyl or (R.sup.3r)

Description

The invention relates to novel alkylated rapamycin derivatives which are pharmaceutically useful, in particular as immunosuppressants.

BACKGROUND OF THE INVENTION

Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus, of the structure represented by formula A:

See, eg, McAlpine, J. B. et al., J. Antibiotics (1991) 44, 688, Schreiber, S. L. et al., J. Am. Chem. Soc. (1991) 113, 7433, U.S. Patent No. 3,929,992. Rapamycin is an extremely potent immunosuppressant and has also been shown to have antitumor and antifungal activity. However, its utility as a pharmaceutical composition is limited by its very low and variable bioavailability as well as its high toxicity. In addition, rapamycin is very insoluble, making it difficult to prepare stable dosage forms.

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SUMMARY OF THE INVENTION

It has now been unexpectedly found that certain novel rapamycin derivatives (novel compounds) have a better pharmacological profile than rapamycin, have better stability and bioavailability, and allow for easier manufacture of dosage forms. The novel compounds are alkylated derivatives of rapamycin having the structure corresponding to the general formula I:

(I) in which

X is (Η, H) or an oxygen atom,

Y is (H, OH) or an oxygen atom, R ¹ and R ² are independently selected from hydrogen, alkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl, acyloxyalkyl, aminoalkyl groups, alkylaminoalkyl groups, alkoxycarbonylaminoalkyl groups, acylaminoalkyl groups, arylsulfonamidoalkyl groups, allyl group, dihydroxyalkylallyl groups, dioxolanylallyl group and hydroxyalkoxyalkyl groups, wherein alk- or alkyl represents a C 1-6 alkyl group having a straight or branched chain, acyl a carboxylic acid residue selected from the group consisting of C 1 -C 6 alkylcarbonyl, nicotinoyl, 2- (N-morpholino) acetyl, 2- (Nimidazolyl) acetyl and 2- (N-methyl-N-piperazinyl) ) an acetyl group , and aryl is phenyl or tolyl, and

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R ⁴ represents a methyl group, or R ⁴ and R ¹ together form an alkylene group having 2 to 6 carbon atoms, provided that R ¹ and R ^{2 are} not both hydrogen.

Preferred novel compounds include the following:

1.40-O-Benzylrapamycin

The second 40-O- ^{(4-hydroxymethyl)} benzyl-rapamycin

3. 40-O- (4 '- (1,2-dihydroxyethyl)) benzylrapamycin

40-O-allylrapamycin

5. 40-O- (3 '- (2,2-dimethyl-1,3-dioxolan-4 (S) -yl) prop-2'-en-1'-yl) rapamycin

6. (2'E, 4'S) -40-O- (4 ', 5'-dihydroxypent-2'-en-1'-yl) rapamycin

7. 40-O- (2-hydroxy) ethoxycarbonylmethylrapamycin

8. 40-O- (2-hydroxy) ethyl-rapamycin

9. 40-O- (3-hydroxy) propylrapamycin

10. 40-O- (6-hydroxy) hexylrapamycin

11.40- O- (2- (2-hydroxy) ethoxy) ethylrapamycin

12. 40-O - ((3S) -2,2-dimethyldioxolan-3-yl) methyl-rapamycin

13.40-O - ((2S) -2,3-dihydroxyprop-1-yl) rapamycin

14. 40-O- (2-acetoxy) ethylrapamycin

15. 40-O- (2-nicotinoyloxy) ethyl-rapamycin

16. 40-O- (2- (N-morpholino) acetoxy) ethylrapamycin

17. 40-O- (2-N-imidazolylacetoxy) ethylrapamycin

18. 40-O- (2- (N-methyl-N ' -piperazinyl) acetoxy) ethylrapamycin

19. 39-0-Desmethyl-39.40-0.0-ethylenrapamycin

(26R) -26-dihydro-40-O- (2-hydroxy) ethyl-rapamycin

21. 28-O-methylrapamycin

22. 40-O- (2-Aminoethyl) rapamycin

23.40- O- (2-acetaminoethyl) rapamycin

24. 40-O- (2-nicotinamidoethyl) rapamycin

25. 40-O- (2- (N-methylimidazo-2'-ylcarboxamido) ethyl) rapamycin

26. 40-O- (2-ethoxycarbonylaminoethyl) rapamycin

27. 40-O- (2-tolylsulfonamidoethyl) rapamycin

28th 40-O- (2- (4 ', 5'-dicarboethoxy-r, 2', ^{3-triazol-r-yl)} ethyl) -rapamycin

The novel compounds for immunosuppressive use are preferably 40-O-substituted rapamycins in which both X and Y represent an oxygen atom. R ² is hydrogen. R ⁴ represents a methyl group and R ¹ has a meaning other than a hydrogen atom, most preferably in which R ¹

Is selected from hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl and aminoalkyl, in particular 40-O- (2-hydroxy) ethylrapamycin, 40-0- (3hydroxy) propylrapamycin, 40-O- (2- (2-hydroxy) ethyl); hydroxy-ethoxy) ethyl-rapamycin and 40-O- (2-acetaminoethyl) rapamycin).

O-substitution at C40 or Ο, dis-disubstitution at C28 and C40 is preferably carried out by the following general procedure: Rapamycin (or dihydro- or deoxorapamycin) is reacted with an organic moiety attached to the eliminated moiety (e.g. RX, where R is organic) a residue, such as an alkyl group, an allyl group or a benzyl group, which is desired as an O-substituent, and X is a eliminated group, for example CCl ₃ C (NH) O or CF ₃ SO ₃ ), under suitable reaction conditions, preferably acidic or neutral conditions, for example in the presence of an acid such as trifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid or their respective pyridinium or substituted pyridinium salts when X is CCl ₃ C (NH) O, or in the presence of a base such as pyridine, substituted pyridine, diisopropylethylamine or pentamethylpiperidine, when X is CF ₃ SO ₃ . Substitution only at C28 is achieved in the same way, but with preliminary protection at C40. Other modifications are contemplated. For example, when the substituent is an allyl group, the isolated, monosubstituted allyl double bond is highly amenable to further modification.

The 9-deoxorapamycin compounds are preferably prepared by reduction of rapamycin using hydrogen sulfide, reaction of rapamycin with diphenyldiselenide attributeylphosphine, or other suitable reduction reaction.

The 26-dihydrorapamicins are preferably prepared by reducing rapamicins or deoxorapamicins from a keto group to a hydroxy group at C 26 by a slight reduction reaction, for example a borohydride reduction reaction.

The novel compounds are particularly suitable for the following purposes:

(a) Treatment and prevention of rejection of transplanted organs or tissues, for example for the treatment of recipients of, eg, transplanted heart, transplanted lung, co-transplanted heart and lung, transplanted liver, kidney, pancreas, transplanted skin or cornea. They are also indicated for the prevention of graft-versus-host reactions, for example following bone marrow transplantation.

(b) Treatment and prevention of autoimmune diseases and inflammatory conditions, in particular inflammatory conditions of setiologies including an autoimmune component such as arthritis (e.g. rheumatoid arthritis, chronic progressive arthritis and deforming arthritis) and rheumatic diseases. Specific autoimmune diseases for which the compounds of the invention may be used include autoimmune hematological disorders (including, for example, haemolytic anemia, aplastic anemia, red blood cell deficiency anemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, scleroderma. Wegener's granulomatosis, dermatomyositosis, chronic active hepatitis, myasthenii gravis, psoriasis. Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (including, eg, ulcerative colitis and Crohn's disease), endocrine ofitalmopathia. Graves' disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), dry keratoconjunctivitis, vemal keratoconjunctivitis, interstitial fibrosis of the lungs, nephrotic glomerulitis or psoriatic arthritis including, but not limited to, idiopathic nephrotic syndrome or nephropathy with minimal changes) and juvenile dermatomyositis.

c) Treatment and prevention of asthma.

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d) Treatment of multi-drug resistance (MDR). The novel compounds suppress P-glycoproteins (Pgp), which are molecules involved in membrane transport in association with MDR. MDR is particularly problematic in cancer and AIDS patients who do not respond to conventional chemotherapy because chemotherapeutic drugs are pumped from the cells by Pgp. The novel compounds are therefore useful for potentiating other chemotherapeutic agents in the treatment and management of multidrug resistance conditions, such as cancer resistant to combination chemotherapy or AIDS resistant to combination chemotherapy.

e) Treatment of proliferative disorders such as tumors, hyperproliferative skin diseases and the like.

f) Treatment of fungal infections.

g) Treatment and prevention of inflammation, in particular by potentiating the effect of steroids.

h) Treatment and prevention of infections, in particular infections caused by pathogens with Mip or Mip-like factors.

i) Treatment of overdose of FK-506, rapamycin, immunosuppressive new compounds and other macrophilin-binding immunosuppressants.

Accordingly, the present invention provides novel compounds for use as novel intermediates or as pharmaceutical compositions, methods of treating or preventing the above disorders by administering an effective amount of the novel compound to a patient in need thereof, using the novel compound in the manufacture of a medicament for treating or preventing the above disorders; pharmaceutical compositions comprising the novel compound in combination or in conjunction with a pharmaceutically acceptable diluent or carrier.

Most of the novel compounds described herein are highly immunosuppressive, especially novel compounds that are O-substituted at C40, and these novel compounds are particularly useful for indications a and b but not for indications i. Novel compounds that are less immunosuppressive, especially those that they are O-substituted only on C28, are particularly useful for indications hai, but are less suitable for indications a or b.

The novel compounds are used by administering a pharmaceutically acceptable form to a patient in need of treatment. Appropriate doses of the novel compounds will, of course, vary depending upon, for example, the condition to be treated (e.g., the type of disease or the nature of resistance), the desired effect and the mode of administration.

In general, however, satisfactory results are obtained after oral administration of doses of the order of from 0.05 to 5 or up to 10 mg / kg / day, for example from 0.1 to 2 or up to 7.5 mg / kg / day given once or in divided doses. 2 to 4 times a day, or after parenteral administration, e.g., intravenously, for example by iv infusion, at doses of the order of from 0.01 to 2.5 to 5 mg / kg / day, for example from 0.05 or 0.1 to 1. mg / kg / day. Thus, a suitable daily dosage for patients is of the order of 500 mg p.o., e.g. of the order of 5 to 100 mg p.o., or of the order of 0.5 to 125 to 250 mg i.v., e.g. of the order of 2.5 to 50 mg.

i.v.

Alternatively, and even preferably, the dosage is adjusted in a patient-specific manner to provide predetermined lowest effective blood levels, e.g., as determined by the RIA technique. Thus, the dosage of the patient may be adjusted to achieve uniform, continuous lowest effective levels, as measured by the RIA technique, of the order of 50 or 150 to 500 or 1000 ng / ml, i.e. analogously to the dosage methods commonly used for cyclosporin immunosuppressive therapy.

The novel compounds may be administered as the sole active ingredient or together with other drugs. For example, in immunosuppressive applications such as prevention and treatment of graft versus host disease, transplant rejection or autoimmune disease, the novel compounds may be used in combination with cyclosporin. FK-506 or immunosuppressive derivatives thereof, corticosteroids, azathioprine, immunosuppressive monoclonal antibodies, eg, monoclonal antibodies to CD3, CD4, CD25, CD28 or CD45 and 7 or other immunomodulatory compounds. For anti-inflammatory applications, the novel compounds may be used in combination with other anti-inflammatory agents, eg, antiviral drugs or antibiotics.

The novel compounds are administered by any conventional route, in particular enterally, e.g. orally, e.g. in the form of drinkable solutions, tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions. Suitable dosage unit forms for oral administration contain, for example, 1 to 50 mg of a compound of the invention, usually 1 to 10 mg. Pharmaceutical compositions containing the novel compounds can be prepared analogously to pharmaceutical compositions containing rapamycin, e.g. as described in EPA 0 041 795, as will be apparent to those skilled in the art.

The pharmacological activity of the novel compounds is demonstrated, for example, by the following tests:

1. Mixed lymphocyte (MLR) response

The response to mixed lymphocytes was originally developed in conjunction with allografts, to determine tissue compatibility between potential organ donors and their recipients, and is one of the best developed in vitro immune response models. Mouse model of MLR, eg as described in Immunological Methods, L. Lefkovits and B. Peris, Eds., Academic Press, Μ. Y. pp. 227-239 (1979) is used to demonstrate the immunosuppressive effect of the novel compounds. Spleen cells (0.5 x 10 ⁶ ) from Balb / c mice (female, 8-10 weeks) are incubated for 5 days together with 0.5 x 10 ⁶ irradiated (2000 rad) or mitomycin C treated spleen cells of CBA mice (female) , 8 to 10 weeks). Irradiated allogeneic cells elicit a proliferative response in the spleen cells of Balb / c mice, which can be measured by incorporating the labeled precursor into DNA. Since stimulator cells are irradiated (or mitomycin C treated), they do not respond to Balb / c mouse cells by proliferation but retain their antigenicity. The antiproliferative effect of the novel compounds on Balb / c mouse cells is measured at various dilutions and the concentration at which 50% inhibition of cell proliferation (IC 50) is obtained. The inhibitory ability of a test sample can be compared to rapamycin and expressed as a relative IC 50 (ie IC _{5 of the} test sample / IC ₅ of rapamycin).

IL-6 mediated proliferation

The ability of the novel compounds to interfere with the growth factor-associated signaling pathways was determined using an interleukin-6 (IL-6) -dependent mouse hybridoma cell line. The experiment was performed in 96-well microtiter plates. 5000 cells / well are cultured in a serum-free environment (as described by H. Schreier and R. Tees in Immunological Methods, I. Lefkovits and B. Pemis, Academic Press 1981, Vol. II, pp. 263-275). supplemented with 1 ng recombinant IL6 / ml. After a 66 hour incubation in the presence or absence of the test sample, cells are pulsed with 1 pCi (3-H) thymidine / cell for an additional 6 hours, harvested and counted with a liquid scintillator. The incorporation of (3-H) thymidine into DNA is correlated with the increase in cell numbers and thus a measure of cell proliferation.

The dilution series of the test samples makes it possible to calculate the concentration at which 50% inhibition of cell proliferation (IC ₅₀ ) occurs. The inhibitory capacity of the test sample can be compared to rapamycin and expressed as relative IC 50 (ie IC _{50 of} test sample / IC ₅ of rapamycin).

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3. Macrophilin binding assay

Rapamycin and the structurally related immunosuppressant FK-506 are known to bind in vivo to macrophilin-12 (also known as FK-506 or FKBP-12 binding protein) and are believed to be related to the immunosuppressive activity of these compounds. The novel compounds also bind strongly to macrophilin-12, as demonstrated by the competitive binding assay.

In this assay, BSA-bound FK-506 is used to cover microtiter wells. Biotinated recombinant human macrophilin-12 (biot-MAP) is allowed to bind in the presence or absence of a test sample to immobilized FK-506. After washing (to remove non-specifically bound macrophilin), bound biot-MAP is evaluated by incubation with streptavidin-alkaline phosphatase conjugate, followed by washing followed by addition of pnitrophenyl phosphate as substrate. The reading is OD at 405 nm. Binding of the test sample to biot-MAP results in a decrease in the amount of biot-MAP bound to FK-506 and thus a decrease in OD405. A series of dilutions of the test sample makes it possible to determine the concentration giving 50% inhibition of biot-MAP binding to immobilized FK-506 (IC50). The inhibitory capacity of the test sample is compared to the IC _{50 of} free FK-506 as a standard and expressed as relative IC ₅₀ (ie IC _{50 of} test sample / IC _{50 of} free FK-506).

4. Localized graft-to-recipient reaction (GvH)

The in vivo efficacy of the novel compounds is demonstrated in a suitable animal model, as described, for example, in Ford et al. Transpantation 10 (1970) 258. Spleen cells (1 x 10 ⁷ ) from 6 week old Wistar / Furth (WF) female rats were injected subcutaneously on day 0 into the left hind paw of approximately 100 female rats (F344 x WF) Fi. g. Animals are continued for 4 consecutive days and sampled lymph nodes on day 7 and weighed. The difference in weight between the two lymph nodes is taken as the basis for evaluating the reaction.

5. Reaction of renal allografts in rat

One kidney from a female Fissher 344 rat is transplanted into the renal vessel of a WF rat recipient after one-sided (left-sided) nephrectomy end to end. The ureter connection is also end to end. Treatment begins on the day of transplantation and continues for 14 days. Seven days after transplantation, contralateral nephrectomy is performed so that the recipient of the transplanted kidney remains solely for kidney function. The survival of the graft recipient is taken as a criterion for graft functionality.

Experimentally induced allergic encephalomyelitis (EAE) in rats

The activity of the novel compounds in EAE is measured, for example, by the procedure described in Levine and Wenk, Amer. J. Path 47 (1965) 61, McFarlin et al. J. Immunol. 113 (1974) 712, Borel, Transplant. and Clin. Immunol. 13 (1981) 3. EAE is a widely established model for multiple sclerosis. Male Wistar rats are injected into the hind paws with a mixture of bovine spinal cord and complete Freund's adjuvant. Symptoms of the disease (paralysis of the tail and both hind legs) usually develop within 16 days. The number of sick animals as well as the time of onset of the disease shall be recorded.

7. Arthritis induced by Freund's adjuvant

Efficacy against experimentally induced arthritis is demonstrated using the procedure described, for example, in Winter and Nuss. Arthrithis and Rheumatism 9 (1966) 394; Billingham and Davies. Handbook of Experimental Pharmacol. (Vane and Ferreira Eds. Springer-Verlag, Berlin) 50/11 (1979) 108-144. OFA and Wistar rats (male or female, 150 g body weight) are injected subcutaneously at the base of the tail or in the hind paw of 0.1. ml of mineral oil containing 0.6 mg of lyophilized, heat-killed Mycobacterium smegmatis. When developing an arthritis model, treatment is started immediately after adjuvant injection (days 1 to 18), already developed

Arthritis model treatment is started on day 14 when secondary inflammation is well developed (14-20.

day). At the end of the experiment, the swelling of the joints is measured using a slide micrometer. ED ₅₀ is an oral dose in mg / kg that reduces swelling (primary or secondary) in half compared to controls.

8. Anti-tumor and MDR activity

The antitumor activity of the novel compounds and their ability to increase the function of the antitumor agents by reducing multicancer resistance is demonstrated, for example, by administering an anticancer agent such as colchicine or etoposide, multidrug resistant cells and drug sensitive cells in vitro or animals that have tumors or infections resistant to combination chemotherapy, with or without co-administration of the novel compounds to be tested and administration of the novel compound itself.

Any suitable cell line and a control (parent) cell line, generated, for example, as described by Ling et al., J. Cell, are used in these assays. Physiol. 83, 103-116 (1974) and Bech-Hansen et al. J. Cell. Physiol. 88, 23-32 (1976). Specific clones selected are the CHR (subclone C553, 2), multidrug resistant (e.g., colchicine resistant) and the parental, sensitive AUX 81 (subclone ABI Sil).

Anti-tumor and anti-MDR activity in vivo is demonstrated, for example, in mice by injection of multi-drug resistant cancer cells and drug-sensitive cancer cells. Ehrlich's ascitic carcinoma (EA) subline resistant to the drug substance DR, VC, AM, ET, TE or CC develops by sequential transfer of EA cells to subsequent generations of BALB / c host mice by the methods described by Slater et al., J. Clin. Invest. 70, 1131 (1982).

Similar results can be obtained using test models of novel compounds of a similar type, eg in vitro, or using test animals infected with drug resistant virus strains and drug-sensitive viral strains, antibiotic resistant bacterial strains (eg penicillin) and bacterial strains sensitive antibiotics, fungal strains antimycotically resistant and sensitive as well as drug resistant protozoal strains, e.g., Plasmodial strains, e.g., naturally occurring Plasmodia falciparum sub-strains, which have acquired resistance to chemotherapeutic, antimalarial drugs.

9. Binding of FKBP

Some of the novel compounds are not immunosuppressive, especially those that are O-substituted at C28 only, such as 28-O-methylrapamycin. This can be demonstrated by standard in vitro assays compared to FK506 and rapamycin. For example, FK506 is known to be a potent inhibitor of IL-2 transcription, as can be demonstrated by an IL-2 gene reporter assay. Rapamycin, although not active in the IL-2 gene reporter assay, has a strong inhibitory effect on IL-6 dependent T cell proliferation. Both compounds are very potent inhibitors of mixed lymphocyte reaction. Non-immunosuppressivity can also be demonstrated in the above-mentioned in vivo medels 1 to 7. However, even those novel compounds that are not immunosuppressive bind to macrophilin, which has certain uses for which non-immunosuppressivity is preferred.

Those novel compounds that bind strongly to macrophilin and which are not immunosuppressive per se can be used in the treatment of an overdose of macrophilin-binding immunosuppressants such as FK506, rapamycin and immunosuppressive novel compounds.

10. Steroid potentiation

The ability of the novel compounds to bind macrophilin also makes them useful for potentiating the effect of corticosteroids. Combination therapy with compounds of the invention and corticosteroids such as

With dexamethasone, a very increased steroid activity is achieved. This can be demonstrated by the MMTV-CAT (chloramphenicol acetyltransferase) tumor reporter assay, eg as described inNING A SPOL., J. Biol. Chem. (1993) 268: 6073. This synergistic effect makes it possible to reduce corticosteroid doses, thereby reducing the risk of side effects in some cases.

11. Inhibition of Mip and Mip-like factors

The novel compounds further bind and block a number of Mip factors (potentiator of macrophage infectivity) and Mip-like factors that are structurally similar to macrophilin. Mip factors and the like are virulent factors produced by a wide variety of pathogens including those of Chlamidia, e.g., Chlamidia trachomatis, Neisseria, e.g. Neisseria meningitidis, and Legionella, e.g. Legionella pneumophilia, and also obligatory parasitic members of the order of Rickettsiales. These factors play a critical role in the development of intracellular infection. The efficacy of the novel compounds in reducing the infectivity of pathogens that produce Mip factors or the like can be illustrated by comparing the infectivity of pathogens in cell culture in the presence or absence of macrolides, eg using the methods described in Lundmose et al., Mol. Microbiol. (1993), 7, 777. These non-immunosuppressive compounds of the invention are preferred for use in this indication because they are not immunosuppressive and therefore do not compromise the body's natural immune defense against pathogens.

The novel compounds are also useful in attempts to detect the presence or amount of macrophilin-binding compounds, eg, in competitive experiments for diagnostic or screening purposes. Accordingly, the present invention also provides the use of the novel compounds as a screening agent to detect the presence of macrophilin-binding compounds in a test solution, e.g., blood, serum or test medium to be inspected. Preferably, the novel compound is immobilized in microtiter wells and then allowed to bind in the presence or absence of a test solution to labeled macrophilin-12 (FKBP-12). Alternatively, FKBP-12 is immobilized in microtiter wells and allowed to bind in the presence and absence of the test solution to a new compound that has been labeled, e.g., fluorine, enzymatically or with a radioactive isotope, such as a new compound that has been O-substituted on C-40. and / or a C28 labeling group. The plates are washed and the amount of bound labeled compound is measured. The amount of macrophilin binding agent in the test solution is roughly inversely proportional to the amount of labeled compound bound. For quantitative analysis, a standard binding curve is constructed using known concentrations of the macrophilin-bound compound.

DETAILED DESCRIPTION OF THE INVENTION

The following examples provide characteristic spectroscopic data to facilitate identification. Peaks not significantly different from rapamycin are not included. Biological data are expressed as relative IC ₅₀ o compared to rapamycin for mixed lymphocyte response (MLR) and IL-6 dependent proliferation assays, and compared to FK-506 in macrophilin binding assay (MBA). A higher IC ₅₀ of lower binding affinity.

Example 1

40-O-benzylrapamycin

To a stirred solution of 183 mg (0.200 mmol) of rapamycin in 2.1 ml of a 2: 1 mixture of cyclohexane and methylene chloride is added 75 μΐ (0.402 mmol) of benzyltrichloroacetimidate, followed by 2.6 μ μ (29 μιηοΐ, 15 mol%) of trifluoromethanesulfonic acid, the mixture immediately

-9GB 283333 B6 turns yellow. After 3 hours the mixture was diluted with ethyl acetate and quenched with 10% aqueous sodium carbonate solution. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic solution was washed with 10% sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography using a 50:50 mixture of hexane and ethyl acetate as eluent to give 40-O-benzylrapamycin as a white amorphous product:

1 H NMR (CDCl ₃ ) δ 0.73 (1H, dd), 1.65 (3H, s), 1.73 (3H, s), 3.12 (4H, sam), 3.33 (3H, s); s), 3.49 (3H, s), 4.15 (1H, dd), 4.65 (1H, d), 4.71 (1H, d), 7.22-7.38 (5H, m) ); MS (FAB) m / z 1026 ((M + Na +)), 972 ((M-OCH ₃ ) +), 954 ((M- (OCH ₃ + H ₂ O)) +).

MBA (rel. IC50) 1.8

IL-6 dep. prol. (rel. IC50) 10

MRL (rel. IC50) 110

Example 2

40-O- (4'-hydroxymethyl) benzylrapamycin

a) 40-O- (4 '- (tert-butyldimethylsilyl) oxymethyl) benzylrapamycin

To a stirred solution of 345 μΐ (2.0 mmol) of triflic anhydride in 5 ml of -78 ° C methylene chloride is added a solution of 504 mg (2.0 mmol) of 4- (tert-butyldimethylsilyl) oxymethylbenzyl alcohol and 820 mg (4 mmol). (0 mmol) of 2,6-di-tert-butyl-4-methylpyridine in 5 ml of methylene chloride. The resulting mixture was warmed to -20 ° C and stirring was continued at this temperature for 0.5 h. The mixture was then re-cooled to -78 ° C and a solution of 914 mg (1.0 mmol) of rapamycin in 5 mL of methylene chloride was added. The mixture was allowed to warm to room temperature overnight and then quenched with 10% sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic solution was washed with saturated brine, dried over sodium sulfate, filtered under reduced pressure and evaporated. The residue was purified by column chromatography on silica gel using hexane-ethyl acetate 50:50 as eluent, to afford 40-O- (4 ^'- (tert-butyldimethylsilyl) oxymethyl) benzyl-rapamycin as a white foam:

MS (FAB) m / z 1170 ((M + Na) +), 1098 ((M- (OCH ₃ + H ₂ O)) +).

b) 40-O- (4'-hydroxymethyl) benzylrapamycin

To a stirred solution of 98 mg (0.093 mmol) of the compound obtained in Example 2 in 2 mL of acetonitrile cooled to 0 ° C was added 0.2 mL of HF-pyridine. The resulting mixture was stirred for 2 hours and quenched with sodium bicarbonate solution, then extracted with ethyl acetate. The organic solution was washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by column chromatography on silica gel using hexane / ethyl acetate (20:80) as eluent to give the title compound as a white foam:

1 H NM (CDCl ₃ ) δ 0.73 (1H, dd), 1.65 (3H, s), 1.74 (3H, s), 3.22 (1H, m), 4.67 (4H, s), m), 7.35 (4H, m), MS (FAB) m / z 1056 ((M + Na) +), 1002 ((M-OCH ₃ ) +), 984 ((M- (OCH ₃ + H)) ₂ O)) +), 966 ((M- (OCH ₃ + 2 H ₂ O)) +), 934 ((M- (OCH ₃ + CH ₃ OH + H 2 O ₂₎₎ +).

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MBA (rel. IC50) 2.7

IL-6 dep. prol. (rel. IC50) 3.9

MRL (rel. IC50) 3

Example 3

40-O- (4 '- (1,2-dihydroxyethyl)) benzyrapamycin

(a) 40-O- (4 '- (2,2-dimethyl-1,3-dioxolan-4-yl)) benzylrapamycin

Dissolve 452 mg (1,24 mmol) of 4- (2,2-dimethyl-1,3-dioxolan-4-yl) benzyltrichloroacetamidate in 10 mL of 1: 1 cyclohexane / methylene chloride, followed by 0.14 mL (0 , 64 mmol) of 2,6-di-tert-butylpyridine and 56 μΐ (0.64 mmol) of trifluoromethanesulfonic acid. To this was added a solution of 587 mg (0.64 mmol) of rapamycin in 2 mL of methylene chloride. The reaction mixture was stirred overnight at room temperature and quenched with sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic solution was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using 50:50 hexane / ethyl acetate as eluent to give 40-O- (4 '- (2,2-dimethyl-3-dioxolan-4-yl)) benzylrapamycin in the form of a white, amorphous, solid product:

1 H NMR (CDCl 3) δ 0.73 (1H, dd), 1.48 (3H, s), 1.55 (3H, s), 1.65 (3H, s), 1.74 (3H, s) ), 3.67 (3H, m), 4.28 (1H, dd), 4.62 (1H, d), 4.69 (1H, d), 5.06 (1H, dd), 7.33 (4H, m); MS (FAB) m / z 1126 ((M + Na) +), 1072 ((M-OCH ₃ ) +), 1054 ((M- (OCH ₃ + H ₂ O)) +), 1014 ((M- (OCH ₃ + CH ₃ COCH ₃ )) *), 996 ((M- (OCH ₃ + H ₂ O + CH ₃ COCH ₃ )) *), 978 ((M- (OCH ₃ +2 H ₂ O + CH ₃₎ COCH ₃₎₎ *).

b) 40-O- (4 '- (1,2-dihydroxyethyl)) benzylrapamycin

To a solution of 90.7 mg (0.08 mmol) of 40-O- (4 '- (2,2-dimethyl-1,3-dioxolan-4-yl)) benzylrapamycin in 4 mL of methanol was added 1 mL of 1 N aqueous HCl. After 2 h, the mixture was quenched with aqueous sodium bicarbonate and extracted twice with ethyl acetate. The organic solution was washed with brine, dried over anhydrous sodium sulfate and evaporated. The residue was purified by silica gel column chromatography using ethyl acetate as eluent. The title compound is obtained in the form of a white foam:

1 HNM (CDCl 3) δ 0.73 (1H, dd), 1.65 (3H, s), 1.74 (3H, s), 3.70 (4H, m), 4.63 (1H, d) 4.69 (1H, d), 4.80 (1H, dd), 7.33 (4H, m); MS (FAB) m / z 1086 ((M + Na) +), 1032 ((M-OCH ₃ ) +), 1014 ((M- (OCH ₃ + H ₂ O)) +), 996 ((M- (OCH ₃ +2 H ₂ O)) *).

MBA (rel IC50) 0.92

IL-6 dep. prol. (rel. IC50) 10.5

MRL (rel. IC50) 22

Example 4

40-O-allylrapamycin

To a stirred solution of 0.33 ml (2.01 mmol) of triflic anhydride in 10 ml of methylene chloride cooled to -78 ° C was slowly added a solution of 0.14 ml (2.06 mmol) of allyl alcohol and 0.42 g (2 mmol). (04 mmol) of 2,6-di-tert-butyl-4-methylpyridine in 5 ml of methylene chloride. The resulting greenish solution was stirred for 1.5 hours and a solution of 915 mg (1.00 mmol) of rapamycin was added

And 0.42 g (2.04 mmol) of 2,6-di-tert-butyl-4-methylpyridine in 5 ml of methylene chloride. Stirring was continued for 0.5 h at -78 ° C and then warmed to room temperature. After an additional 1 hour, the mixture was quenched with aqueous sodium bicarbonate solution and the layers separated. The aqueous layer was extracted twice with ethyl acetate. The combined organic solution was washed with aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The resulting green oil was purified by silica gel column chromatography using 60:40 hexane: ethyl acetate to give the title compound as a colorless amorphous solid:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.65 (3H, s), 1.74 (3H, s), 3.05 (IH, m), 4.13 (2H, bd), 5.14 (2H, m), 5.27 (2H, m), 5.92 (2H, m); MS (FAB) m / z 976 ((M + Na) +), 922 ((M-OCH ₃ ) +), 904 ((M- (OCH ₃ + H ₂ O)) +), 886 ((M- (OCH ₃ +2 H ₂ O) *), 872 ((M- (2CH ₃ OH + OH)) *), 854 ((M (OCH ₃ + CH ₃ OH + 2 H ₂ O)) *).

MBA rel. IC501

IL-6. prol. (rel. IC50) 8

MRL (rel. IC50) 260

Example 5

40-O- (3 '- (2,2-dimethyl-1,3-dioxolane-4 (S) -yl-prop-2'-en-1'-yl) rapamycin)

To a stirred solution of 0.64 g (4.00 mmol) of E- (4S) -4,5-O, O-isopropylidenepent-2-en-1,4,5-triol and 1.26 g (6.00 mmol) of 2 6-Di-tert-butyl-4-methylpyridine in 20 ml of methylene chloride cooled to -78 ° C was added 0.82 ml (5 mmol) of triflic anhydride. The resulting mixture was stirred at this temperature for 2 hours and a solution of 1.82 g (2.00 mmol) of rapamycin and 1.26 g (6.00 mmol) of 2,6-di-tert-butyl-methylpyridine in 5 ml methylene chloride. The mixture was allowed to gradually warm to room temperature overnight and then quenched with aqueous sodium bicarbonate solution. The layers were separated and the aqueous layer was extracted three times with ethyl acetate. The organic solution was washed with aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using a 40:60 mixture of hexane and ethyl acetate to give the title compound as a white solid:

1 HNMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.38 (3H, s), 1.42 (3H, s), 1.65 (3H, s), 1.73 (3H, s) ), 3.06 (1H, m), 3.58 (2H, m), 4.08 (IH, dd), 4.15 (2H, m), 4.52 (IH, dd), 5.72 (1H, m), 5.88 (1H, m); MS (FAB) m / z 1076 ((M + Na) +), 1022 ((M-CH ₃ ) +), 1004 ((M-OCH ₃ + H ₂ O)) +), 964 ((MOH ₃ +) CH ₃ COCH ₃ ), 946 ((M-OCH ₃ +2 H ₂ O + CH ₃ COCH ₃ )) *).

MBA (rel IC50) 0.64

IL-6 dep. prol. (rel. IC50) 11

MRL (rel. IC50) 8

Example 6 (2'E, 4'S) -40-O- (4,5'-dihydroxypent-2'-en-1'-yl) rapamycin

The conditions described in Example 3, step b), applied to the compound obtained in the previous example, followed by purification by silica gel column chromatography using a mixture of ethyl acetate and methanol as the eluent, provided the title compound as a white foam:

- 12CZ 283333 B6 1 H NMR (CDCl ₃ ) δ 0.68 (1H, dd), 3.04 (1H, m), 4.18 (5H, m), 5.75 (1H, dd), 5, 88 (1 H, m); MS (FAB) m / z 1036 ((M + Na) +), 1013 (M +), 995 ((MH ₂ O) +), 982 ((M-OCH ₃ ) +), 964 ((M (OCH ₃₎₎ 3 + H ₂ O) *), 946 ((M- (OCH ₃ +2 H ₂ O)) *), 832 ((M- (2CH ₃ OH + OH)) *), 914 ((M (OCH ₃ )) + CH 3 OH + 2 H ₂ O) *).

MBA (rel. IC50) 1.7

11-6 dep. prol. (rel. IC50) 12

MRL (rel. IC50) 3.5

Example 7

40-O- (2-hydroxy) ethoxycarbonylmethylrapamycin

a) 40-O- (2- (tert-butyldimethylsilyl) oxy) ethoxycarbonylmethylrapamycin

To a stirred solution of 2.74 g (3.00 mmol) of rapamycin and 30 mg (0.06 mmol) of rhodium acetate dihydrate in 30 mL of methylene chloride was added a solution of 0.38 mL (3.60 mmol) of 2- (tert-butyldimethyl silyl). of oxyethyldiazoacetate in 10 ml of methylene chloride over 5 hours. After the addition was complete, stirring was continued for an additional hour, after which the reaction was quenched with 1N aqueous HCl. The layers were separated and the aqueous layer was extracted with ethyl acetate. The combined organic solution was washed with aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using a 40:60 mixture of hexane and ethyl acetate as eluent to give 4Q-O- (2- (tert-butyldimethylsilyl) oxy) ethoxycarbonylmethyl-rapamycin:

1 H NMR (CDCl 3) δ 0.06 (6H, s), 0.68 (1H, dd), 0.88 (9H, s), 1.64 (3H, s), 1.73 (3H, s) 1.12 (5H, sam), 3.81 (2H, dd), 4.19 (2H, dd), 4.32 (2H, s); MS (FAB) m / z 1152 ((M + Na) +), 1080 ((M (OCH ₃ + H ₂ O)) +).

b) 40-O- (2-hydroxy) ethoxycarbonylmethylrapamycin

To a stirred solution of 81 mg (0.07 mmol) of 40-O- (2- (tert-butyldimethylsilyl) oxy) ethoxycarbonylmethylrapamycin in 1.5 mL of acetonitrile cooled to 0 ° C was added 0.15 mL of HFpyridine. After 2 hours, the reaction is quenched with aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate. The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue is purified by chromatography using ethyl acetate as the eluent to give the title compound as a white solid:

1 H NMR (CDCl 3) δ 0.70 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.13 (5H, sam), 3.85 (3H, m) 4.25 (5H, m); MS (FAB) m / z 1038 ((M + Na) +), 984 ((M-OCH ₃ ) +), 966 ((M- (OCH ₃ + H ₂ O)) +, 948 ((M- ( OCH ₃ +2 H ₂ O)) *).

MBA (rel. IC50) 4

IL-6 dep. prol. (rel IC50) 9.7

MRL (rel IC50) 2.1

- 13GB 283333 B6

Example 8

40-O- (2-hydroxy) ethyl-rapamycin

a) 40-O- (2- (tert-butyldimethylsilyl) oxy) ethylrapamycin

A solution of 9.14 g (10 mmol) of rapamycin and 4.7 mL (40 mmol) of 2,6-lutidine in 30 mL of toluene was heated to 60 ° C and treated with a solution of 6.17 g (20 mmol) of 2- of (t-butyldimethylsilyl) oxyethyl triflate and 2.35 ml (20 mmol) of 2,6-lutidine in 20 ml of toluene. The mixture was stirred for 1.5 hours. Two portions of a solution of 3.08 g (10 mmol) of triflate and 1.2 ml (10 mmol) of 2,6-lutidine in 10 ml of toluene are added over 1.5 hours. After the last batch was added, stirring was continued at 60 ° C for 2 hours and the resulting brown suspension was filtered. The filtrate was diluted with ethyl acetate and washed with aqueous sodium bicarbonate solution and brine. The organic solution was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using a 40:60 hexane-ethyl acetate mixture as eluent to give 40-O- (2- (tert-butyldimethylsilyl) oxy) ethylrapamycin as a white solid:

1 H NMR (CDCl 3) δ 0.06 (6H, s), 0.72 (1H, dd), 0.90 (9H, s), 1.65 (3H, s), 1.75 (3H, s) 1.02, 3.02 (1H, m), 3.63 (3H, m), 3.72 (3H, m); MS (FAB) m / z 1094 ((M + Na) +), 1022 ((M- (OCH ₃ + H ₂ O)) +).

b) 40-O- (2-hydroxy) ethylrapamycin

To a stirred solution of 4.5 g (4.2 mmol) of 40-O- (2- (tert-butyldimethylsilyl) oxy) ethylrapamycin in 20 mL of methanol cooled to 0 ° C was added 2 mL of 1N HCl. This solution was stirred for 2 hours and neutralized with aqueous sodium bicarbonate. The mixture was extracted with three portions of ethyl acetate. The organic solution was washed with aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. Purification by silica gel column chromatography eluting with ethyl acetate gave the title compound as a white solid:

1 H NMR (CDCl 3) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.13 (5H, sam), 3.52-3.91 (8H, m); MS (FAB) m / z 980 ((M + Na) +), 926 ((M-OCH ₃ ) +), 908 ((M- (OCH ₃ + H ₂ O))), 890 ((M (OCH) ₃ +2 H ₂ O)) +), 876 (M- (2CH ₃ OH + OH)) *) 858 ((M- (OCH ₃ + CH ₃ OH + H 2 O ₂₎₎ +).

MBA rel. IC50) 2.2

IL-6 dep. prol. (rel. IC50) 2.8

MRL (rel. IC50) 3.4

Example 9

40-O- (3-hydroxy) propylrapamycin

a) 40-O- (3- (tert-butyldimethylsilyl) oxypropylrapamycin)

Using the same procedure as described in Example 8, step a), using 3- (tert-butyldimethylsilyl) oxyprop-1-yltriflate affords 40-O- (3- (tert-butylmethylsilyl) oxy) propylrapamycin:

1 H NMR (CDCl ₃ δ 0.05 (6H, s), 0.72 (1H, dd), 0.90 (9H, s), 1.65 (3H, s), 1.74 (3H, s) 1.77 (2H, m), 3.03 (1H, m), 3.52 3.73 (7H, m); MS (FAB) m / z 1108 ((M + Na) +), 1036 ((M (OCH ₃ + H ₂ O)) +).

- 14GB 283333 B6

b) 40-O- (3-hydroxy) propylrapamycin

Treatment of the compound prepared in step a) under the conditions described in Example 8, step b, affords the title compound:

1 H NM (CDCl ₃ ) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 1.80 (2H, m), 3.05 (1H, m), 3.55 3.91 (8H, m), MS (FAB) m / z 994 ((M + Na) +), 940 ((M-OCH 3) +), 922 ((M- (OCH _{3) 3)} + H ₂₀ O) *), 904 ((M- (OCH ₃ +2 H ₂ O)) *), 872 ((M- (OCH ₃ + CH ₃ OH + 2 H ₂ O)) *).

MBA (rel. IC50) 1.6

IL-6 dep. prol. (rel. IC50) 2.7

MRL (rel. IC50) 11

Example 10

40-O- (6-hydroxy) hexylrapamycin

a) 40-O- (6- (tert-butyldimethylsilyl) oxy) hexylrapamycin

Using the same procedure as described in Example 8, step a), using 6- (tert-butyldimethylsilyl) oxyhex-1-yltriflate affords 40- (6- (tert-butyldimethylsilyl) oxy) hexylrapamycin:

MS (FAB) m / z 1150 ((M-Na) < + >).

b) 40-O- (6-hydroxy) hexylrapamycin

Treatment of the compound prepared in step a) under the conditions described in Example 8, step b, affords the title compound:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.38 (2H, m), 1.57 (4H, m), 1.65 (3H, s), 1.74 (3H, s), 3.02 (1H, m), 3.49 3.72 (8H, m): MS (FAB) m / z 1036 ((M + Na) +), 982 ((M-OCH ₃ ) +) ), 964 ((M (OCH ₃ + H ₂ O)) *), 946 ((M- (OCH _{3 +} H ₂ O)) *), 914 ((M- (OCH ₃ + CH ₃ OH + 2 H) ₂ O)) *).

MBA (rel IC50) 0.8

IL-6 dep. prol. (rel. IC50) 8.5

MRL (rel. IC50) 18

Example 11

40-O- (2- (2-hydroxy) ethoxy) ethylrapamycin

a) 40-O- (2- (tert-butyldimethylsilyl) oxyethoxy) ethylrapamycin

Using the same procedure as described in Example 8, step a), using 2- (2- (tert-Butyldimethylsilyl) oxyethoxy) ethyltriflate affords 40-O- (2- (tert-butyldimethylsilyl) oxyothoxy) ethylrapamycin:

1 H NMR (CDCl ₃ ) δ 0.06 (6H, s), 0.71 (1H, dd), 0.88 (9H, s), 1.65 (3H, s), 1.74 (3H, s) 3.07 (1H, m), 3.51-3.79 (11H, m); MS (FAB) m / z 1138 ((M + Na) +), 1115 (M +), 1097 ((MH ₂ O) +), 1084 ((M-OCH ₃ ) +), 1066 ((M- ( OCH ₃ + H ₂ O) *), 1048 ((M- (OCH ₃ +2 H ₂ O)) *), 1034 ((M (2CH ₃ OH + OH)) *), 1016 ((M- ( OCH ₃ + CH ₃ OH + H ₂ O 2)) +).

- 15GB 283333 B6

b) 40-O- (2- (2-hydroxy) ethoxy) ethylrapamycin

Treatment of the compound prepared in step a) under the conditions described in Example 8 step b) affords the title compound:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.65 (3H, s), 1.74 (3H, s), 3.05 (IH, m), 3.51-3, 77 (1H, m); MS (FAB) m / z 1024 ((M + Na) +), 1001 (M +), 983 ((MH ₂ O) +), 970 ((M-OCH ₃₎ +) 952 ((M (OCH ₃ +)) H ₂ O) *, 934 ((M-COCH ₃ +2 H ₂ O)) *), 920 ((M- (2CH ₃ OH + OH)) *), 902 ((M (OCH 3 + CH 3 OH + 2 H) ₂ O)) *).

MB A (rel. IC50) 1.2

IL-6 dep. prol. (rel. IC50) 3.2

MRL (rel. IC50) 2

Example 12

40-O - ((3S) -2,2-dimethyldioxolan-3-yl) methyl-rapamycin

Using the same procedure as described in Example 8, step a), the title compound was obtained using glycerol acetone triflate:

1 H NMR (CDCl 3) δ 0.72 (1H, dd), 1.36 (3H, s), 1.42 (3H, s), 1.65 (3H, s), 1.75 (3H, s) 3.06 (1H, m), 3.55 (2H, m), 3.69 (3H, m), 4.06 (1H, dd), 4.26 (1H, m); MS (FAB) m / z 1050 ((M + Na) +), 996 ((M-OCH ₃₎ +), 978 ((M- (OCH ₃ + H ₂ O)) +), 960 ((M- ( OCH ₃ +2 H ₂ O)) *).

MBA (rel. IC50) 0.9

IL-6 dep. prol. (rel. IC50) 8

MRL (rel. IC50) 290

Example 13

40-0 - ((2S) -2,3-dihydroxyprop-1-yl) rapamycin

Treatment of the compound prepared in the previous example under the conditions described in Example 3 affords the title compound:

1 H NMR (CDCl 3) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.07 (IH, m), 3.68 (8H, s), m); MS (FAB) m / z 1010 ((M + Na) +), 956 ((M-OCH 3) +), 938 ((M- (OCH 3 + H ₂ O) +)), 920 ((M- (OCH ₃ +)) 2 H ₂ O)) +), 888 ((M4OCH3 + CH3OH + 2 H ₂ O)) +).

MBA (rel. IC50) 0.67

IL-6 dep. prol. (rel. IC50) 9

MRL (rel. IC50) 10

Example 14

40-O- (2-acetoxy) ethylrapamycin

To a stirred solution of 40 mg of O-hydroxyethylrapamycin (53 mg, 0.055 mmol) in methylene chloride (2 mL) cooled to 0 ° C was added pyridine (0.2 mL), followed by 0.02 mL (0.281 mmol).

- 16EN 283333 B6 acetyl chloride. The mixture was stirred for 3 hours and diluted with ethyl acetate, then washed with aqueous sodium bicarbonate, cold 1N HCl, and again aqueous sodium bicarbonate. The organic solution was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using 30:70 hexane / ethyl acetate to give the title compound as a white solid:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 2.08 (3H, s), 3.07 (1H, s), m), 3.78 (2H, dd), 4.20 (2H, dd); MS (FAB) m / z 1022 ((M + Na) +), 999 (M +), 982 ((M-OH) +), 968 ((MOH ₃ ) +), 950 ((M (OCH ₃ +)) H ₂ O) *), 932 ((M- (OCH ₃ +2 H ₂ O)) *), 918 ((M- (2CH ₃ OH + OH)) *), 900 ((M- (OCH ₃ + CH ₃ OH + 2 H ₂ O) *).

MBA (rel. IC50) 2

IL-6 dep. prol. (rel. IC50) 7.6

MRL (rel. IC50) 3.6

Example 15

40-O- (2-nicotinoyloxy) ethyl-rapamycin

Using the same procedure as described in the previous example, using nicotinoyl chloride hydrochloride, the title compound is obtained:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.65 (3H, s), 1.75 (3H, s), 3.07 (1H, m), 3.94 (2H, s), dd), 4.49 (2H, t), 7.39 (1H, dd), 8.31 (1H, ddd)), 8.78 (1H, ddd), 9.24 (1H, dd), MS ( FAB) m / z 1085 ((M + Na) +), 1063 ((M + H) +), 1045 ((M + OH) +), 1031 ((M-OCH ₃ + H ₂ O)) +.

MBA (rel. IC50) 11

IL-6 dep. prol. (rel. IC50) 6.9

MRL (rel. IC50) 5

Example 16

40-O- (2- (N-morpholino) acetoxy) ethylrapamycin

(a) 40-O- (2-bromoacetoxy) ethylrapamycin

Using the same procedure as described in Example 14, using bromoacetyl chloride, 40-0 (2-bromoacetoxy) ethyl lrapamycin was obtained:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.67 (3H, s), 1.76 (3H, s), 3.03 (1H, m), 3.82 (2H, s), m), 3.87 (2H, s), 4.31 (2H, m); MS (FAB) m / z 1100, 1102 ((M + Ma) +), 1077 (M +), 1061 ((MH ₂ O) +), 1046, 1048 ((M-OCH ₃ ) +), 1028, 1030 ((M- (OCH ₃ + H ₂ O)) *), 1012 ((M- (OCH ₃ + H ₂ O)) +), 996 ((M- (2CH ₃ OH + OH)) *) 980 ((M (OCH ₃ + CH ₃ OH + 2 H ₂ O)) *.

b) 40-O- (2- (N-morpholino) acetoxy) ethylrapamycin

To a stirred solution of 54 mg (0.05 mmol) of 40-O- (2-bromoacetoxy) ethylrapamycin in 0.2 N, N-dimethylformamide cooled to -45 ° C was added a solution of 0.022 mL (0.25 mmol) of morpholine in 0 2 ml of Ν, Ν-dimethylformamide and the resulting mixture was stirred at this temperature for 1 hour, after which an aqueous solution of sodium bicarbonate was added. This mixture was extracted three times

- 17GB 283333 B6 with ethyl acetate. The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using a 95: 5 mixture of ethyl acetate and methanol as the eluent to give the title compound as an amorphous white product:

1 H NMR (CDCl 3) δ 0.72 (1H, dd), 1.67 (3H, s), 1.76 (3H, s), 2.60 (3H, m), 3.07 (1H, m) 1.24 (2H, s), 3.78 (8H, m), 4.27 (2H, t); MS (FAB) m / z 1107 ((M + Na +)), 1085 ((M + H) +), 1067 ((MOH) +), 1053 ((M-OCH ₃ ) +), 1035 ((M- (OCH ₃ + H ₂ O)) +).

MBA (rel. IC50) 1.3

IL-6 dep. prol. (rel. IC50) 4

MRL (rel. IC50) 3.5

Example 17

40-O- (2-N-imidazolylacetoxy) ethylrapamycin

In the same manner as described in Example 16, step b), using the imidazole, the title compound was obtained:

1 H NMR (CDCl ₃ ) δ 0.72 (1H, dd), 1.67 (3H, s), 1.78 (3H, s), 3.06 (1H, m), 3.80 (2H, m), 4.32 (2H, m), 4.73 (2H, s), 6.97 (1H, dd), 7.09 (1H, dd), 7.52 (1H, dd); MS (FAB) m / z 1066 ((M + H) +), 1048 ((M-OH) +), 1034 ((M-OCH ₃ ) +), 1016 ((M- (OCH ₃ + H ₂ O)) ) *).

MBA (rel. IC50) 1

IL-6 dep. prol. (rel. IC50) 7.6

MRL (rel. IC50) 3.4

Example 18

40-O- (2- (N-methyl-N'-piperazinyl) acetoxy) ethylrapamycin

Using the same procedure as described in Example 16, step b), using N-methylpiperazine, the title compound was obtained:

1 H NMR (CDCl 3) δ 0.72 (1H, dd), 1.67 (3H, s), 1.77 (3H, s), 2.78 (4H, sam), 3.02 (4H, bs) 1.08, 3.08 (1H, m), 3.32 (2H, s), 3.80 (2H, dd), 4.27 (2H, t); MS (FAB) m / z 1098 ((M + H) +), 1066 ((MOCH ₃ ) +).

MBA (rel. IC50) 2.6

IL-6 dep. prol. (rel. IC50) 10.3

MRL (rel. IC50) 5

Example 19

39-0-desmethyl-39.40-0.0-ethylenrapamycin

To a stirred solution of 48-mg (0.05 mmol) of 40-O-hydroxyethylrapamycin and 0.023 ml (0.20 mmol) of 2,6-lutidine in 0.5 ml of methylene chloride cooled to -20 ° C was added 0.008 ml ( 0.05 mmol) of triflic anhydride. The mixture was stirred at this temperature for 2 hours and then left

- 18GB 283333 B6 to warm to room temperature and stir for an additional hour. The reaction was quenched by the addition of aqueous sodium bicarbonate solution and the resulting mixture was extracted with three portions of ethyl acetate. The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using 30:70 hexane / ethyl acetate as eluent to give the title compound as a white solid:

1 H NMR (CDCl 3) δ 1.66 (3H, s), 1.75 (3H, s), 3.14 (3H, s), 3.35 (3H, s), 3.76 (4H, s) ); MS (FAB) m / z 948 ((M + Na) +), 925 (M +), 908 ((M-OH) +), 894 ((M-OCH ₃ ) +), 876 ((M- ( OCH ₃ + H ₂ O *), 858 ((M- (OCH ₃ +2 H ₂ O)) *), 844 ((M- (2CH ₃ OH + OH)) *), 826 (M- (OCH ₃ + CH ₂ OH + 2 H ₂ O) *).

MBA (rel.IC50) 1.6

IL-6 dep. prol. (rel. IC50) 22.9

MRL (rel. IC50) 16

Example 20 (26 R) -26-Dihydro-40-O- (2-hydroxy) ethyl-rapamycin

a) (26R) -26-Dihydro-40-O- (2- (tert-butyldimethylsilyloxy)) ethylrapamycin

315 mg (1.2 mmol) of tetramethylammonium triacetoxyborohydride are dissolved in 4.5 ml of 2: 1 acetonitrile: acetic acid. The resulting solution was stirred at room temperature for 1 hour and cooled to -35 ° C, then 161 mg (0.15 mmol) of 40-O- (2- (tert-butyldimethylsilyl) oxy) ethylrapamycin was added. The resulting mixture was stirred overnight at the same temperature and quenched with aqueous sodium bicarbonate solution. The mixture was extracted with three portions of ethyl acetate. The organic solution was washed with aqueous sodium bicarbonate solution, two portions of 30% aqueous Rochelle salt solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by column chromatography on silica gel using hexane / ethyl acetate (40:60) as eluent to give the title compound as a white solid:

1 H NMR (CDCl ₃ ) δ 0.06 (6H, s), 0.73 (1H, dd), 0.90 (9H, s), 1.64 (3H, s), 1.67 (3H, s), s), 3.02 (1H, m), 3.15 (1H, m), 3.64 (3H, m), 3.71 (2H, dd), 3.91 (1H, s); MS (FAB) m / z 1096 ((M + Na) +), 1041 ((M-HOCH ₃ ) +), 1024 ((M- (OCH ₃ + H ₂ O)) +), 1006 ((M-) (OCH ₃ +2 H ₂ O)) *), 974 ((M (OCH ₃ + CH ₃ OH + 2 H ₂ O)) *).

b) (26 R) -26-dihydro-40-O- (2-hydroxy) ethyl-rapamycin

Treatment of the compound prepared in step a) under the conditions described in Example 8 step b) affords the title compound:

1 H NMR (CDCl ₃ ) δ 0.75 (1H, dd), 1.66 (3H, s), 1.70 (3H, s), 3.18 (1H, m), 3.52-3, 84 (7H, m), MS (FAB) m / z 982 ((M + Na) +), 928 ((M-OCH ₃ ) +), 910 ((M- (OCH ₃ + H ₂ O))) * ), 892 ((M (OCH ₃ +2 H ₂ O)) *).

MBA (rel. IC50) 3.9

IL-6 dep. prol. (rel. IC50) 53

MRL (rel. IC50) 18

- 19GB 283333 B6

Example 21

8-O-methyl-lrapamycin

To a stirred solution of 103 mg (0.1 mmol) of 40-O-TBS-rapamycin (obtained by silylating rapamycin with 1 equivalent of TBS triflate in methylene chloride in the presence of 2 equivalents of 2,6-glutidine at 0 ° C) in 0.5 mL of methylene chloride is added 85 8 mg (0.40 mmol) of proton fungus, followed by 44 mg (0.30 mmol) of trimethyloxonium tetrafluoroborate. The resulting brown heterogeneous mixture was stirred overnight, quenched with aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic solution was washed with 1N HCl, aqueous sodium bicarbonate and brine, then dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography using a 60:40 mixture of hexane and ethyl acetate as eluent to give 40-tert-butyldimethylsilyl-28-O-methylrapamycin. This compound was desilylated under the conditions described in Example 10, step b) to give the title compound as a white solid after PTLC with ethyl acetate:

1 H NMR (CDCl ₃ ) δ 0.70 (1H, dd), 1.68 (6H, 2s), 2.95 (IH, m), 3.13 (3H, s), 3.14 (3H, 2H); s), 3.28 (3H, s), 3.41 (3H, s); MS (FAB) m / z 950 ((M + Na) +), 927 (M +), 909 ((MH ₂ O) +), 896 ((MOCH ₃ ) +), 878 ((M- (OCH _{3) +)} + H ₂ O) *), 864 ((M- (OCH ₃ + CH ₃ OH)) *), 846 ((M- (2CH ₃ OH + OH)) *), 832 ((M- (OCH ₃₎ + 2CH ₃ OH + ₁ ), 814 ((M- (3CH ₃ OH + OH)) +).

MBA (rel IC50) 1.58

IL-6 dep. prol. (rel. IC50) 1240

MRL (rel. IC50) 1300

Example 22

40-O- (2-aminoethyl) rapamycin

(a) 40-O- (2-bromoethyl) rapamycin

A solution of 914 mg of rapamycin in 5 ml of toluene containing 0.64 ml of 2,6-lutidine and 1.28 g of 2-bromoethyl triflate was heated at 65 ° C for 18 hours. The reaction mixture was then cooled to room temperature, poured onto 20 mL of saturated sodium bicarbonate solution and extracted with 3 x 20 mL of ethyl acetate. The organic phases are dried over sodium carbonate and the solvent is distilled off under reduced pressure on a rotary evaporator. The residue is chromatographed on 100 g of silica gel, eluting with a 3: 2 mixture of hexane and ethyl acetate. There was thus obtained 40-O- (2-bromoethyl) rapamycin as an amorphous solid product:

MS (FAB) m / z 1044 and 1042 (100%, M + Na); 972 and 970 (55%, M- (MeOH + H ₂ O)). 1 HNMR (CDCl ₃ ) d: 0.72 (1H, q, J = 12 Hz); 3.13 (3 H, s); 3.33 (3 H, s); 3.45 (3 H, s); 3.9 (4 H, m); 4.78 (1H, s)

b) 40-O- (2-azidoethyl) rapamycin

A solution of 2.4 g of 40-O- (2-bromoethyl) rapamycin in 40 ml of Ν, Ν-dimethylformamide was treated with 0.19 g of sodium azide at room temperature. After 2 hours, the mixture is poured onto 100 ml of saturated sodium bicarbonate solution and extracted with 3 x 100 ml of ethyl acetate. The organic phases were combined, dried over sodium sulfate and the solvent was distilled off under reduced pressure. The crude product is purified by chromatography on silica gel, eluting with hexane-ethyl acetate. There was thus obtained 40-O- (2-azidoethyl) rapamycin:

-20GB 283333 B6

MS (FAB): 1005 (100%, Ma + Na); 951 (24%, M-MeOH); 933 (57%, M - (MeOH + H ₂ O)

c) 40-O- (2-aminoethyl) rapamycin

To a solution of 230 mg of 40-O- (azidoethyl) rapamycin in 3 ml of a 5: 1 mixture of tetrahydrofuran and water was added 307 mg of triphenylphosphine at room temperature. The reaction mixture turns yellow. After 7 hours, the reaction mixture is chromatographed on silica gel, eluting with a 50:50: 0.5 mixture of ethyl acetate, methanol and acetic acid. The title product is obtained in the form of acetate:

MS (FAB) m / z 979 (45%, M + Na); 957 (100%, M); 925 (63%, M-MeOH); 907 (25%, M (MeOH + H ₂ O)

MBA (rel. IC50) 0.7

IL-6 dep. prol. (rel. IC50) 10

Example 23

40-O- (2-acetaminoethyl) rapamycin

To a solution of 101 mg of 40-O- (2-aminoethyl) rapamycin acetate in 2 mL of tetrahydrofuran was added 0.02 pyridine and 0.07 mL of acetyl chloride. The reaction mixture is kept at room temperature for 18 hours and then poured onto 7 ml of saturated sodium bicarbonate solution. The aqueous phase is extracted three times with 5 ml of ethyl acetate, the organic phases are combined and dried over sodium sulfate. The solvent was evaporated and the residue was chromatographed on 10 g of silica gel, eluting first with ethyl acetate and then with a 50:50: 0.5 mixture of ethyl acetate, methanol and acetic acid. This yields the following product in the title:

MS (FAB) m / z 1021 (20%, M + Na); 967 (28%, M-MeOH); 949 (100%, M- (MeOH + H ₂ O) 1 H-NMR (CDCl ₃ ) d: 0.71 (1H, q, J = 12 Hz); 1.98 (3H, s); 3.13 ( 3H, s), 3.34 (3H, s), 3.44 (3H, s), 4.75 (1H, s)

MBA (rel. IC50) 1.1

IL-6 dep. prol. (rel IC50): 2.3

Example 24

40-O- (2-nicotinamidoethyl) rapamycin

101 mg of 40-O- (2-aminoethyl) rapamycin acetate are dissolved in 5 ml of ethyl acetate and extracted twice with saturated sodium bicarbonate solution. The organic phase is dried over sodium sulphate and the solvent is distilled off. The residue is dissolved in 2 ml of tetrahydrofuran and treated with 22 mg of Ν, Ν'-dicyclohexylcarbodiimide and 15 mg of nicotinic acid. After 15 hours at room temperature, the reaction mixture was evaporated and the residue was chromatographed on silica gel, eluting with ethyl acetate followed by a 9: 1 mixture of ethyl acetate and methanol. This yields the following product in the title:

MS (FAB) m / z 1084 (80%, M + Na); 1062 (40%, MH); 1038 (100%, M-MeOH); 1012 (50%, M- (MeOH + H ₂ O)

H-NM (CDCl3) d: 0.72 (1H, q, J = 12 Hz); 3.13 (3 H, s); 3.33 (3 H, s); 3.37 (3 H, s); 7.39 (1H, dd, J = 6Hz, J = 8Hz), 8.19 (1H, d, J = 8Hz); 8.75 (1H, d, J = 6Hz); 9.04 (IH, broad s)

-21 GB 283333 B6

MBA (rel. IC50) 1.2

IL-6 dep. prol. (rel. IC50) 2.8

Example 25

40-O- (2- (N-methylimidazo-2'-ylcarboxamido) ethyl) rapamycin

To a solution of 30 mg of N-methylimidazole-2-carboxylic acid in 1 ml of dimethylformamide was added 58 mg of Ν, Ν-dicyclohexylcarbodiimide and 58 mg of hydroxybenztriazole. After 2 hours, 150 mg of 40-O- (2-aminoethyl) rapamycin was added and the reaction mixture was stirred at room temperature for 18 hours. The suspension is then filtered, the filtrate is diluted with 5 ml of ethyl acetate and washed twice with 2 ml of a saturated aqueous sodium bicarbonate solution. The organic phase is dried over sodium sulphate and the solvent is evaporated off under reduced pressure. The residue is chromatographed on silica gel, eluting with hexane / ethyl acetate (1: 4) and then with ethyl acetate. This yields the following product in the title:

MS (FAB) m / z 1087 (36%, M + Na); 1065 (57%, MH); 1033 (100%, M-MeOH); 1015 (46%, M - (MeOH + H ₂ O))

H-NMR (CDC1 ₃₎ d: 0.72 (1H, q, J = 12 Hz); 3.13 (3 H, s); 3.33 (3 H, s); 3.46 (3 H, s); 4.03 (3 H, s); 6.93 (1H, broad s); 7.78 (1 H, m)

MBA (rel. IC50) 1.1

IL-6 dep. prol. (rel. IC50) 7

Example 26

40-O- (2-ethoxycarbonylaminoethyl) rapamycin

A solution of 200 mg of 40-O- (2-azidoethyl) rapamycin in 3 mL of 5: 1 tetrahydrofuran / water was treated with 267 mg of triphenylphosphine at room temperature for 7 hours. Then add 0.4 ml of pyridine, followed by 194 μΐ of ethyl chloroformate. After 2 hours, the reaction mixture is poured onto 5 mL of ethyl acetate and washed successively with 10 mL of saturated aqueous sodium bicarbonate, 5 mL of water and 5 mL of 10% citric acid. The organic phase is dried over sodium sulphate and the solvent is distilled off. The residue is chromatographed over 20 g of silica gel, eluting with ethyl acetate and then with a 9: 1 mixture of ethyl acetate and methanol. This yields the following product in the title:

MS (FAB) m / z 1051 (35%, M + Na); 997 (30%, M-MeOH); 979 (100%, M- (MeOH + H ₂ O)) 1 H-NMR (CDCl ₃ ) d: 0.71 (1H, q, J = 12 Hz); 1.24 (3H, t, J = 8Hz); 3.13 (3 H, s); 3.34 (3 H, s); 3.43 (3 H, s); 4.10 (2H, q, J = 8Hz); 5.48 (1 H, m)

MBA (rel. IC50) 1.1

IL-6 dep. prol. (rel. IC50) 1.7

-22EN 283333 B6

Example 27

40-O- (2-tolylsulfonamidoethyl) rapamycin

A solution of 200 mg of 40-O- (2-aminoethyl) rapamycin in 3 ml of tetrahydrofuran was treated with 0.4 ml of pyridine and 390 mg of tosyl chloride while stirring at room temperature for 12 hours. The solution is then poured into 5 ml of saturated bicarbonate solution and the aqueous phase is extracted twice with 5 ml of ethyl acetate. The combined organic phases were washed with 5 ml of 10% citric acid and 5 ml of water. After drying over sodium sulfate, the solvent is distilled off and the residue is chromatographed on 20 g of silica gel, eluting with hexane / ethyl acetate (1: 1). The title product is obtained in the form of a white foam:

MS (FAB) m / z 1133 (100%, M + Na); 1078 (25%, M-MeOH); 1061 (85%, M- (MeOH + H ₂ O)) H-NM (CDCl ₃ ) d: 0.68 (1H, q, J = 12Hz); 2.43 (3 H, s); 3.13 (3 H, s); 3.35 (3 H, s); 3.41 (3 H, s); 4.76 (1 H, s); 5.85 (1H, t, J = 6 Hz); 7.30 (2H, d, J = 8Hz); 7.75 (2H, d, J = 8 Hz).

MBA (rel. IC50) 15.9

IL-6 dep. prol. (rel. IC50) 14

Example 28

40-O- (2- (4 ', 5'-dicarboethoxy-1,2,3'-triazol-1-yl) ethyl) rapamycin mg of 40-O- (2-azidoethyl) rapamycin and 32 mg of diethylacetylenedicarboxylate are suspended in 0.5 ml toluene and heated at 65 ° C for 5 hours. The reaction mixture was then cooled to room temperature, loaded onto 10 g of silica gel and eluted with hexane-ethyl acetate 1: 1. This yields the following product in the title:

MS (FAB) m / z 1175 (20%, M + Na); 1121 (15%, M-MeOH); 1103 (60%, M- (MeOH + H ₂ O)) 1 H-NMR (CDCl ₃ ) d: 0.62 (1H, q, J = 12 Hz); 1.40 (3H, t, J = 8Hz); 1.42 (3H, t, J = 8Hz); 3.13 (3 H, s); 3.25 (3 H, s); 3.33 (3 H, s)

MBA (rel. IC50) 2.7

IL-6 dep. prol. (rel. IC50) 12

The foregoing examples can also be performed using 9-deoxorapamycin, 26-dihydrorapamycin or 9-deoxo-, 26-dihydrorapamycin as the starting material instead of rapamycin. Alternatively and preferably, as described, for example, in Example 20, the rapamycin compounds of the above examples may be hydrogenated or reduced, optionally using suitable protecting groups. New methods for reducing the keto group to C9 or hydrogenating the keto group to C26 are also described.

Example 29

Removal of the keto group on C9

At room temperature, a stream of hydrogen sulfide is passed through a stirred solution of 3.2 g (3.5 mmol) of rapamycin in 50 ml of pyridine and 2.5 ml of Ν, Ν-dimethylformamide. The initially colorless solution turns yellow. After two hours, the hydrogen sulfide feed was stopped and stirring was continued for five days. During this time, the solution gradually turns orange. Thin layer chromatography and high performance liquid chromatography confirm complete consumption of the starting material and the presence of a single new compound. The solution was purged with nitrogen for one hour and evaporated under reduced pressure.

-23 GB 283333 B6

The residue is taken up in ethyl acetate, washed three times with cold 1N HCl solution, saturated sodium bicarbonate solution and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue is taken up in ether and the precipitated sulfur is filtered off. Evaporation of the ether solution followed by silica gel chromatography (10: 4: 1 CH ₂ Cl ₂ / i-Pr ₂ O / MeOH) gave 9-deoxorapamycin as a colorless foam. The identity of the product is confirmed by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and / or infrared spectroscopy (IR). The following characteristic physical values were observed for 9-deoxorapamycin:

1 H NMR (CDCl ₃ ) 61.61 (3H, d, J = 1Hz, C 17 -CH 3), 1.76 (3H, d, J = 1.2Hz, C 29 -CH ₃ ), 2.42 ( 1H, d, J = 14.5 Hz, H-9), 2.74 (1H, d, J = 14.5 Hz, H-9), 3.13 (3H, s, OCH _3-CI6) 3.5 (3H, s, _C27-OCH3), 3.40 (3H, s, _C39-OCH3), 5.40 (1H, d, J = 10Hz, H-30), 5.57 (1 H δ d, δ = 8.6 Hz, J ₂ = 15 Hz, H-22), 5.96 (1H, d, J = 9 Hz, H-18), 6.09 (1H, d, J = 1) 7 Hz, 10-OH), 6.15 (1H, dd, £ = 10 Hz, J ₂ = L5 Hz, H-21), 6.37 (1H, dd, £ = 1.5 Hz, J ₂ = 5 Hz, H 19), 6.38 (1H, J = 9.5 Hz, H-20), ¹³ C NM (CDCl ₃ ) δ 38.5 (C-9), 98.0 (C-10) 170.7 (CI), 173.0 (C-8), 208.8 (C-32), 216.9 (C-26) MS (FAB) m / z 922 (M + Na +)), 899 (M *), 881 ((MH ₂ O) *), 868 ((M-OCH ₃ ) *), 850 ((M (H ₂ O + OCH ₃ )) *)

IR (major peaks) (cm ^-1 ) 987, 1086, 1193, 1453, 1616, 1717, 1739, 3443

MBA (rel. IC50) 1

MRL (rel. IC50) 14

IL-6 dep. prol. (rel. IC50) 9

Example 30

Dihydrogenation of the keto group to C26

To a stirred solution of 421 mg (1.6 mmol) of tetramethylammonium triacetoxyborohydride in 2 mL of acetonitrile was added 2 mL of acetic acid. The resulting mixture was stirred at room temperature for 30 minutes and then cooled to -35 ° C. A solution of 180 mg (0.2 mmol) of 9-deoxorapamycin in 1 ml of acetonitrile was added at this temperature and the resulting mixture was stirred for 24 hours. A saturated aqueous solution of sodium potassium tartrate was added to the mixture and allowed to warm to room temperature. Stirring was continued until both layers were clear, then ethyl acetate was added. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The resulting organic solution was washed once with 10% sodium bicarbonate solution and twice with saturated brine, then dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (90:10 AcOEt-hexane). The starting material in this case was 9-deoxorapamycin, the resulting compound being deoxorapamycin, and 26-dihydrorapamycin was obtained as a colorless foam with the following characteristic spectroscopic values:

1 H NMR (CDCl ₃ (major isomer) δ 9 (3H, d, J = 6.9 Hz, CHCH ₃ ), 0.93 (3H, d, J = 6.9 Hz, CHCH ₃ ), 1.00 (3H, d, J = 6.9 Hz, CH _3), 1.07 (3H, d, J = 6.9 Hz, CH _3), 1.17 (3H, d, J = 6.9 Hz, CH _3), 1.61 (3H, d, J = 1 Hz, _C17-CH3), 1.73 (3H, d, J = 2 Hz, _C29-CH3), 2.43 (1H, dd, J = 4.1 and 16.0 Hz, H-33), 2.46 (1H, d, J = 13.8 Hz, H-9), 2.58 (1H, m, H-25), 2.77 (1H, d, J = 13.8Hz, H-9), 2.82 (1H, dd, J = 8.3 and 16.0 Hz, H-33), 3.17 (1H, dd, J = 4.1 and 9.2 Hz, H-27), 3.61 (2H, m, H-14 and H28), 5.19 (1H, ddd, J = 4.1, 4.6 and 8) 3 Hz, H-34), 5.49 (1H, broad d, J = 5.0 Hz, H-2), 5.56 (1H, d, J = 9.1 Hz, H-30), 5.75 (1H, dd, J = 6.9 and 14.7 Hz, H-22), 5.76 (1H, s, 10-OH), 5.99 (1H, broad d, J = 9, 2 Hz, H-18), 6.10 (1 H, m, H-21), 6.36 (2 H, m, H-19 and H-20);

-24GB 283333 B6

MS (FAB) m / z 924 ((M + Na)), 852 ((M- (H ₂ O + CH ₃ O)) *)

MBA (rel. IC50) 47

MRL (rel. IC50) 134

IL-6 dep. prol. (rel. IC50) 78

26-dihydrorapamycin is prepared in the same manner using rapamycin instead of 9-deoxorapamycin. This product has the following characteristic values:

¹³ C-NMR (CDCl ₃ ) (major isomer) d = 208.3 (C-32), 194.0 (C-9), 169.3 (Cl), 166.6 (C-8), 140, 9 (C-22), 136.5 (C-29), 136.2 (C-17), 133.5 (C-20), 129.1 (C-21), 128.7 (C-18) ), 126.2 (C-30), 125.3 (C-19), 98.6 (C-10), 84.4 (C-39), 83.9 (C-16), 81.6 (C-27), 75.4 (C-34), 74.3 (C-28), 73.9 (C-40), 72.9 (C-26), 67.4 (C-14) , 59.1 (27-OCH ₃ ), 56.6 (39-OCH ₃ ), 55.9 (16-OCH ₃ ), 51.3 (C2), 46.8 (C-31), 44.3 (C-6), 40.4 (C-33), 40.4 (C-25), 39.5 (C-24), 38.8 (C-15), 38.0 (C-36) , 34.3 (C-23), 34.2 (C-38), 33.5 (C-1), 33.3 (C-37), 33.2 (C-35), 31.5 (C) -42), 31.3 (C-41), 30.9 (C-13), 27.1 (C-12), 27.0 (C-3), 25.2 (C-5), 21 4 (23-CH ₃ ), 20.7 (C-4), 17.3 (11-CH ₃ ), 16.1 (31-CH ₃ ), 15.9 (35-CH ₃ ), 14, 4 (25-CH ₃ ), 14.2 (29-CH ₃ ), 10.3 (17-CH ₃ ).

MS (FAB) m / z: 884 (M-OCH ₃ , 35%), 866 (M- (OCH ₃ + H ₂ O), 100%), 848 (M- (OCH ₃ + 2H ₂ O), 40 %)

MBA (rel. IC50) 1.7

MRL (rel. IC50) 1

IL-6 dep. prol. (rel. IC50) 7.5

Claims (28)

PATENT CLAIMS O-alkylated rapamycin derivatives of general formula I (I) -25GB 283333 B6 in which X is (Η, H) or an oxygen atom, Y is (H, OH) or an oxygen atom, R ¹ and R ² are independently selected from hydrogen, alkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl, acyloxyalkyl, aminoalkyl groups, alkylaminoalkyl groups, alkoxycarbonylaminoalkyl groups, acylaminoalkyl groups, arylsulfonamidoalkyl groups, allyl group, dihydroxyalkylallyl groups, dioxolanylallyl group and hydroxyalkoxyalkyl groups, wherein alk- or alkyl represents a C 1-6 alkyl group having a straight or branched chain, acyl a carboxylic acid residue selected from the group consisting of C 1 -C 6 alkylcarbonyl, nicotinoyl, 2- (N-morpholino) acetyl, 2- (Nimidazolyl) acetyl and 2- (N-methyl-N-piperazinyl) ) an acetyl group , and aryl is phenyl or tolyl, and R ⁴ represents a methyl group, or R ⁴ and R ¹ together form an alkylene group having 2 to 6 carbon atoms, provided that R ¹ and R ^{2 are} not both hydrogen. O-alkylated rapamycin derivatives of formula (I) according to claim 1, selected from the group consisting of 1.40-O-benzylrapamycin, 2. 40-O- (4-hydroxymethyl) benzylrapamycin 3. 40-O- [4 '- (1,2-dihydroxyethyl)] benzylrapamycin; 4. 40-O-allylrapamycin 5. 40-O- [3 '- (2,2-dimethyl-1,3-dioxolan-4 (S) -yl) prop-2'-en-1'-yl] rapamycin, 6. (2'E, 4'S) -40-O- (4 ', 5'-dihydroxypent-2'-en-1'-yl) rapamycin, 7. 40-O- (2-hydroxy) ethoxycarbonylmethylrapamycin 8. 40-O- (2-hydroxy) ethylrapamycin 9. 40-O- (3-hydroxy) propylrapamycin 10. 40-O- (6-hydroxy) hexylrapamycin 11.40- O- [2- (2-hydroxy) ethoxy] ethyirapamycin, 12. 40-O - [(3S) -2,2-dimethyldioxolan-3-yl] methyl-rapamycin, 13. 40-O - [(2S) -2,3-dihydroxyprop-1-yl] rapamycin; 14. 40-O- (2-acetoxy) ethylrapamycin 15. 40-O- (2-nicotinoyloxy) ethyl-rapamycin, 16. 40-O- [2- (N-morpholino) acetoxy] ethylrapamycin -26GB 283333 B6 17. 40-O- (2-N-imidazolylacetoxy) ethylrapamycin 18. 40-O- [2- (N-methyl-N'-piperazinyl) acetoxy] ethylrapamycin; 19. 39-0-desmethyl-39,40-0,0-ethylenrapamycin 20. (26R) -26-dihydro-40-O- (2-hydroxy) ethylrapamycin 21.28-O-methylrapamycin, 22. 40-O- (2-aminoethyl) rapamycin 23.40- O- (2-acetaminoethyl) rapamycin, 24.40- O- (2-nicotinamidoethyl) rapamycin, 25.40- O- (2- (N-methylimidazo-2'-ylcarboxamido) ethyl) rapamycin, 26. 40-O- (2-ethoxycarbonylaminoethyl) rapamycin 27. 40-O- (2-tolylsulfonamidoethyl) rapamycin a 28. 40-O- [2- (4 ', 5'-Diethoxycarbonyl-1,2', 3'-triazol-1'-yl) ethyl] rapamycin. O-alkylated rapamycin derivatives of the general formula I according to claim 1, in which X and Y are each oxygen, R ² is hydrogen, R ⁴ is methyl and R ¹ is other than hydrogen. O-alkylated rapamycin derivatives of formula I according to claim 3, wherein R ¹ is selected from the group consisting of hydroxyalkyl groups, hydroxyalkoxyalkyl groups, acylaminoalkyl groups and aminoalkyl groups, wherein acyl and alkyl are as defined above. 5. An O-alkylated rapamycin derivative of the formula I as claimed in claim 4 which is 40-O- (2-hydroxy) ethyl-lrapamycin. A process for the preparation of O-alkylated rapamycin derivatives of formula I according to any one of claims 1 to 5, comprising (i) reacting rapamycin, deoxorapamycin or dihydrorapamycin, optionally in an O-protected form, with an organic radical of R ¹ or R ² as defined in claim 1, optionally in protected form, bound to a leaving group (X) such that (a) X is CC1 ₃ (NH) O and the reaction conducted in the presence of an acid, or (b) X is CF ₃ SO ₃ - and the reaction is carried out in the presence of a base, and (ii) optionally reducing and / or, if necessary, removing the protecting group from the product. An O-alkylated rapamycin derivative of the general formula I according to any one of claims 1 to 5 for use as a medicament. A pharmaceutical composition comprising an O-alkylated rapamycin derivative of the Formula I according to any one of claims 1 to 5 together with a pharmaceutically acceptable diluent or carrier. O-alkylated rapamycin derivatives of formula I according to any one of claims 1 to 5 for use in the manufacture of a medicament for the treatment or prevention of any of the following conditions: (I) autoimmune disease, (ii) allograft rejection, (iii) graft-versus-host disease, (iv) asthma, (v) multi-drug resistance, (vi) tumors or hyperproliferative disorders, or (vii) fungal infections, (viii) inflammation, (ix) infection by pathogens with Mip or Mip-like factors, or (x) an overdose of macrophilin-binding immunosuppressive agents.